Inhibition of s6 kinaze activity for the treatment of insulin resistance

ABSTRACT

This invention provides screening methods for agents effective in treating insulin resistance through specific inhibition of S6 kinase 1 activity. Also provided are methods of treating insulin resistance by administering an effective amount of an inhibitor specific for S6 kinase 1.

The current invention relates to insulin resistance and diabetes, inparticular to the treatment of insulin resistance or diabetes withmodulators of S6 kinase (S6K) activity.

BACKGROUND OF THE INVENTION

Type II diabetes is the most common form of diabetes in the Westernworld and is strongly linked to obesity—over 80% of sufferers are obese.In patients with Type II diabetes, insulin is less able to promote theuptake of glucose into muscle and fat, a state termed insulinresistance. The molecular basis by which obesity leads to impairedinsulin action are not well understood.

Insulin normally acts to maintain glucose homeostasis by regulating itsown production and secretion by pancreatic beta cells, and bycontrolling glucose utilization in peripheral tissues. Recent studieshave implicated the mTOR/S6 kinase signal transduction pathway in thisprocess. S6 kinase is a kinase that phosphorylates the ribosomalprotein, S6. In particular, S6K1 (also known as p70/p85 S6 kinase)deficient mice are mildly glucose intolerant (hyperglycaemic) andhypoinsulinemic, not due to a lesion in glucose sensing or insulinproduction, but to a reduction in pancreatic endocrine mass, which isaccounted by a selective decrease in beta-cell. S6K1 deficient micemaintain normal fasting glucose levels, suggesting they may behypersensitive to insulin in their peripheral tissues (Pende et al.,2000, Nature, 408, 994-997).

This phenotype is reminiscent of a form of preclinical type 2 diabetesmellitus, where protein malnutrition-induced hypoinsulinaemiapredisposes individuals to glucose intolerance. A limited period ofprotein malnutrition in rats also leads to mild glucose intolerancearising from a persistent decrease in β cell size and insulin secretion,an effect partially attenuated by mild insulin hypersensitivity inperipheral tissues (Swenne et al., 1992, Diabetologia 35, 939-945;Swenne et al., J. Endocrinol. 118, 295-302; Grace et al., 1990, DiabetesMetab. 16, 484-491 (1990).

S6K1-deficient mice are viable and fertile but exhibit a conspicuousreduction in body size during embryogenesis, an effect mostly overcomeby adulthood. A comparison of homozygous mutant mice at 3.5 weeks of agedemonstrated that the weights of all organs were proportional to thereduction in body weight. The small size of the homozygous mutant miceis consistent with a defect in translational capacity (Shima et al.,1998, EMBO J., 17, 6649-6659). As S6K1 deficient mice reach maturity,the difference in weight that they display at birth, as compared to wildtype mice, diminishes from 20% to 15%. Such mice might accumulate fatand become insulin resistant as a function of age and an increase indietary fat, as would wild type mice.

The role of S6 kinase activity in insulin resistance has been addressedin human studies. Insulin-sensitive and insulin-resistant, non-diabeticPima Indians were treated with insulin over 2 hours. Although basallevels of S6 kinase activity were similar for both groups, S6 kinase wasactivated only three-fold in insulin resistant individuals compared tofive fold in insulin-sensitive individuals, suggesting an impaired S6kinase activity in insulin resistant individuals.

S6K activity has previously been implicated in cancer and angiogenesis.WO93/19752 describes the use of rapamycin and its derivatives asinhibitors of p70 S6 kinase and their use to inhibit proliferation orthe immune response of a cell. US 2003/0083284 describes antisensecompounds for the inhibition of expression of p70 S6 kinase (referringto both the 70 kDa and nuclear, 85 kDa isoforms). The antisense nucleicacids are proposed to be potentially useful in treating infections,inflammation and tumor formation, as well as metabolic disorders.US2003/0143656 proposes that compounds capable of increasing theactivity of p70 S6K may be useful in treating diabetes or obesity, ormay be useful in inhibiting apoptosis.

Attoub et al. (2001, Faseb J., 14, 2329-2338) show that rapamycin blocksleptin function, in particular leptin-induced invasion of cells intocollagen gels (as a model of carcinogenesis). Leptin therapy has beenused to promote weight loss while preserving lean mass in obese patientswith congenital leptin deficiency, suggesting leptin in the treatment ofobesity or diabetes.

There remains a need to provide new targets and to develop newmedicaments for the treatment of insulin resistance, in particular TypeII diabetes (also referred to as non-insulin dependent diabetesmellitus, NIDDM) and this invention meets that need.

SUMMARY OF THE INVENTION

In accordance with a first aspect of the invention, a method ofidentifying an agent effective in treating insulin resistance isprovided, the method comprising the steps of: i) incubating S6 kinasewith a compound; ii) detecting S6 kinase activity; and iii) determininga compound-induced modulation in the S6 kinase activity relative to whenthe compound is absent, wherein an alteration of the S6 kinase activityin the presence of the compound is indicative of an agent effective intreating insulin resistance. The compound -induced modulation ispreferably independent of an effect of mammalian Target of Rapamycin(mTOR) activity. In one embodiment, the modulation is inhibition of S6kinase 1 activity. S6 kinase activity can be conveniently assayed usingS6 as a substrate and is easily amenable to high throughput assays.

Also provided by the invention are methods of screening for an agenteffective in treating insulin resistance, comprising contactingtranscriptionally active cellular components with a nucleic acidencoding an S6K gene operably linked to a promoter sequence or an S6Kpromoter sequence operably linked to a reporter gene in the presence ofat least one compound; and detecting an effect of the compound on S6kinase expression or S6 kinase promoter activity, wherein detection of adecrease or an increase in S6 kinase expression or promoter activity isindicative of an agent effective in treating insulin resistance. Suchassays can be cell-based assays, where the transcriptionally activecellular components and nucleic acid is present in a cell. In preferredembodiments, the S6 kinase is S6 kinase 1.

The invention further provides methods of identifying an agent effectivein treating insulin resistance, comprising: providing a non-human animalcomprising an S6 kinase gene; administering a compound to the non-humananimal; and determining whether insulin resistance is affected relativeto when the compound is absent. The S6 kinase gene can be from the sameor different species as the transgenic animal (for example a mousecomprising an S6 kinase gene derived from human sequences).

Also encompassed are agents identified by the methods of the invention.

In a further aspect, a method for reducing adipocyte size is provided,comprising contacting an adipocyte with an effective amount of an S6kinase 1 inhibitor.

In yet another aspect, methods for treating a insulin resistance,comprising administering to a subject a pharmaceutically effectiveamount of an S6 kinase modulator are provided. The S6 modulator can bean S6K1 inhibitor.

Thus, also provided are specific modulators of S6 kinase for themanufacture of a medicament for the treatment or prophylactic treatmentof insulin resistance, such as a selective inhibitor of S6 kinase 1.

Similarly, also provided are modulators of S6 kinase activity for use intreating insulin resistance, such as a selective inhibitor of S6 kinase1.

In a further aspect of the invention, a method of diagnosing apredisposition to insulin resistance or diabetes is provided,comprising: obtaining a sample from an individual, detecting the levelof S6 kinase activity, preferably S6 kinase 1 activity, in the sampleand correlating a change in S6 kinase activity in the sample whencompared to a normal control value or range of values with apredisposition to insulin resistance or diabetes. For Example, anincrease in S6 kinase 1 activity when compared to a normal control valueor range of values is indicative of a predisposition to insulinresistance or diabetes.

Also provided are methods of evaluating treatments for insulinresistance, the method comprising administering a therapeutic agent to anon-human animal comprising an S6 kinase gene, in particular S6K1, anddetermining the effect of the agent on insulin resistance.

DETAILED DESCRIPTION OF THE INVENTION

There remains a need for more effective therapies to treat insulinresistance and Type II diabetes in individuals, particularly in obeseindividuals. The present inventors have discovered that in contrast topreviously published studies, S6K1 activation results in insulinresistance thereby providing S6K1 as a pharmaceutical target fortreating diabetes and related maladies through inhibition of S6K1activity. Although mTOR (mammalian target of rapamycin, whichphosphorylates and activates S6 kinase) can be targeted to modulate S6K1activity, direct targeting of S6K1 avoids more general side-effects ofinhibiting mTOR activity and provides more specificity for the treatmentof patients with insulin resistance. In particular, mTOR is known toactivate both S6K1 and S6K2, whereas the present inventors have foundthat the selective inhibition of S6K1 is desirable.

Accordingly, the present invention provides a method of identifying anagent effective in treating insulin resistance or diabetes (inparticular high fat diet or obesity induced conditions), based on themodulation of S6 kinase activity, in particular S6 kinase 1 activity.Typically such a method will comprise the steps of incubating S6 kinase(or a functional equivalent or derivative thereof) with a compound;detecting S6 kinase activity; and determining the compound-inducedmodulation in the S6 kinase activity relative to when the compound isabsent. An alteration of the S6 kinase activity in the presence of thecompound is indicative of an agent effective in treating insulinresistance or diabetes. A control assay in the absence of the compoundcan be run in parallel.

Unless otherwise clear from the context, “S6K” or “S6 kinase” is usedherein to encompass both S6K1 and S6K2 (see, for example GenebankAccession No. M57428, AJ007938, AB019245, NM003952 and relatedsequences), although S6K1 is preferred. Exemplary functional equivalents(variants) or derivatives of S6K include molecules where S6K iscovalently modified by substitution, chemical, enzymatic, or otherappropriate means with a moiety other than a naturally occurring aminoacid.

Generally speaking such variants will be substantially homologous to the‘wild type’ or other sequence specified herein i.e. will share sequencesimilarity or identity therewith. Similarity or identity may be at thenucleotide sequence and/or encoded amino acid sequence level, and willpreferably, be at least about 50%, 60%, or 70%, or 80%, most preferablyat least about 90%, 95%, 96%, 97%, 98% or 99%. Sequence comparisons maybe made using FASTA and FASTP (see Pearson & Lipman, 1988. Methods inEnzymology 183: 63-98). Parameters are preferably set, using the defaultmatrix, as follows: Gapopen (penalty for the first residue in a gap):−12 for proteins/−16 for DNA;

Gapext (penalty for additional residues in a gap): −2 for proteins/−4for DNA; KTUP word length: 2 for proteins/6 for DNA. Analysis forsimilarity can also be carried out using hybridisation. One commonformula for calculating the stringency conditions required to achievehybridization between nucleic acid molecules of a specified sequencehomology is: Tm=81.5oC+16.6 Log [Na+]+0.41(% G+C)−0.63(%formamide)−600/#bp in duplex (Molecular Cloning: a Laboratory Manual:2nd edition, Sambrook et al., 1989, Cold Spring Harbor LaboratoryPress).

Variants that retain common structural features can be fragments of S6K,in particular fragments maintaining catalytic activity or isoformspecific characteristics. For example, the carboxy- terminal sequencesof S6K1 and S6K2 exhibit only about 20% identity and it therefore may beuseful to include such isoform-specific features in fragments whenisoform-specific assays are desired. Similarly, S6K1 can bephosphorylated at T447, which is absent in S6K2 providing an alternativeor additional isoform-specific feature. Preferably, fragments will bebetween 50 and 350 amino acids in length.

Variants of S6K also comprise mutants thereof, which may contain aminoacid deletions, additions or substitutions, subject to the requirementto maintain at least one feature characteristic of S6K, preferablycatalytic activity and/or isoform-specific features as described above.Thus, conservative amino acid substitutions may be made substantiallywithout altering the nature of S6K, as may truncations. Additions andsubstitutions may moreover be made to the fragments of S6K used in thescreening methods of the invention, in particular those enhancing S6Kcatalytic activity or providing some other desirable property. Forexample T389, T229 and S371 in mouse S6K1 (also known as p70S6K) arehomologous to T389, T238 and S380 in Drosophila p70S6K. T389 isparticularly indicated for mutation to an acidic amino acid residue inorder to produce a constitutively active kinase. Fusion proteins withthe S6K or S6K fragments referred to above may also be desirable.

The screening assays of the invention are not limited to any particularmethod of determining S6 kinase activity. S6 kinase assays are wellknown in the art (see for example U.S. Pat. No. 6,372,467, which isherein incorporated by reference in its entirety). Briefly, S6 kinasewill be incubated with a suitable substrate, such as S6, in a bufferallowing phosphorylation of S6. Phosphorylation of the substrate can bedetected using a labelled phosphate group, such as the use of theradioactive label ³²P present as the ATP source in the buffer.Alternatively, antibodies specific for the phosphorylated products ofS6K catalytic activity can be used to detect activity. As will beapparent to those of ordinary skill in the art, the assays are easilyamenable to high through-put technologies using robotics and automatedprocesses.

Alternatively, the S6 kinase activity can be assayed using a syntheticsubstrate, such as those comprising Arg- (Arg)-Arg-X-X-Ser-X (e.g.,KRRRLASLAA or KRRRLSSLRASTSKSESSQK) (Flowtow and Thomas (1992) J. Biol.Chem. 267: 3074-3078.

S6K activity can also be assayed by detecting downstream targets of thekinase. For example S6K is known to affect transcription and translationof specific targets, such as genes with polypyrimidine tracts (5′TOPs)and ribosomal genes. (Fumagalli S, Thomas G. (1999) Ribosmal Protein S6Phosphorylation and Signal Transduction. In: Translational Control. Eds.Hershey, J, Mathews, M, Sonenberg, N. Cold Spring Harbor Press. pp695-717).

Thus, in accordance with a further aspect of the invention, a method isprovided for screening an agent effective in treating insulinresistance, by identifying compounds that modulate expression of an S6Kgene or a gene expressed under the control of S6K regulatory sequences.

Such methods comprise contacting transcriptionally active cellularcomponents, preferably in a cell, with a nucleic acid encoding an S6Kgene operably linked to a promoter sequence or an S6K promoter sequence(or other S6K regulatory regions allowing expression of the reportergene) operably linked to a reporter gene in the presence of at least onecompound; and detecting an effect of the compound on expression of thecoding region, be it S6 kinase expression or reporter gene expression.Expression of S6 kinase can be detected at the transcript level (forexample by hybridization using specific probes or PCR) or at the proteinlevel (for example using an antibody). A decrease or an increase in S6kinase expression or promoter activity is indicative of an agenteffective in treating insulin resistance. Such assays can be cell-basedassays, where the transcriptionally active cellular components andnucleic acid is present in a cell, although in vitro transcriptionassays are also well known in the art. In preferred embodiments, the S6kinase is S6 kinase 1.

The reporter gene encodes any molecule capable of providing a detectablechange. Such reporter molecules include fluorescent moieties (e.g.,fluorescent proteins, such as, cyan fluorescent protein, CFP; yellowfluorescent protein, YFP; blue fluorescent protein, BFP; or greenfluorescent protein, GFP; all available commercially, Clontech LivingColors User Manual, antigens, reporter enzymes and the like. Reporterenzymes include, but are not limited to, the following:beta-galactosidase, glucosidases, chloramphenicol acetyltransferase(CAT), glucoronidases, luciferase, peroxidases, phosphatases,oxidoreductases, dehydrogenases, transferases, isomerases, kinases,reductases, deaminases, catalases and urease. In selecting a reportermolecule to be used in the presently claimed method, the reportermolecule itself should not be inactivated by any putative agent or othercomponent present in the screening assay, including inactivation by anyprotease activity present in the assay mixture. The selection of anappropriate reporter molecule will be readily apparent to those skilledin the art.

The nucleic acid will typically be provided in a vector allowingreplication in one or more selected host cells, as is well known for avariety of bacteria, yeast, and mammalian cells. For example variousviral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful forcloning vectors in mammalian cells. The vector may, for example be inthe form of a plasmid, cosmid, viral particle, phage, or any othersuitable vector or construct which can be taken up by a cell and used toexpress the sequence of interest or reporter gene.

Expression vectors usually contain a promoter operably linked to theprotein-encoding nucleic acid sequence of interest, so as to direct mRNAsynthesis. Promoters recognized by a variety of potential host cells arewell known, as are the S6K1 and S6K2 promoters (upstream regulatorysequences). “Operably linked” means joined as part of the same nucleicacid molecule, suitably positioned and oriented for transcription to beinitiated from the promoter. DNA operably linked to a promoter is “undertranscriptional control” of the promoter. Transcription from vectors inmammalian host cells is controlled, for example by promoters obtainedfrom the genomes of viruses such as polyoma virus, fowipox virus,adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus40 (SV40), from heterologous mammalian promoters, e.g. the actinpromoter or an immunoglobulin promoter, and from heat-shock promoters,provided such promoters are compatible with the host cell systems.Expression vectors of the invention may also contain one or moreselection genes, such as genes conferring resistance to antibiotics orother toxins.

The methods of the invention may therefore further include introducingthe nucleic acid into a host cell. The introduction, which may(particularly for in vitro introduction) be generally referred towithout limitation as “transformation”, may employ any availabletechnique. For eukaryotic cells, suitable techniques may include calciumphosphate transfection, DEAE-Dextran, electroporation, liposome-mediatedtransfection and transduction using retrovirus or other virus, e.g.vaccinia, as is well known in the art. See, for example Keown et al.,Methods in Enzymology, 185:527 537 (1990) and Mansour et al., Nature336:348-352 (1988).

Host cells transfected or transformed with expression or cloning vectorsdescribed herein may be cultured in conventional nutrient media. Theculture conditions, such as media, temperature, pH and the like, can beselected by the skilled artisan without undue experimentation. Ingeneral, principles, protocols, and practical techniques for maximizingthe productivity of cell cultures can be found in “Mammalian CellBiotechnology: a Practical Approach”, M. Butler, ed. JRL Press, (1991)and Sambrook et al, supra.

Assays specific for S6K1 can also be designed by detecting binding ofspecific proteins, such as nerabin to the C-terminal domain of S6K1, asis well known in the art (Burnett P E, Blackshaw S, Lai M M, Qureshi IA, Burnett A F, Sabatini D M, Snyder S H. Neurabin is a synaptic proteinlinking p70 S6 kinase and the neuronal cytoskeleton. Proc Natl Acad SciUSA. 1998 Jul. 7;95(14):8351-6).

Thus, in another embodiment of the invention the inhibition of theinteraction of S6K1 with a binding partner is assessed. This maycomprise (i) contacting S6K1 with a binding partner thereof in thepresence and absence of a test substance; and (ii) determining whetherthe presence of a test substance inhibits the interaction between S6K1and its binding partner.

Methods for assessing the interaction between a polypeptide and abinding partner may be any of the methods known to those skilled in theart and are disclosed here. Any of these methods can be used to assesswhether a test substance inhibits the interaction between a polypeptide(in this case S6K1) and a binding partner.

In one embodiment, assays are those based upon S6K1 and its interactionwith neurabin and comprise the step of determining whether the testsubstance inhibits the interaction between S6K1 and neurabin. This maybe achieved by detecting the physical association between S6K1 and itsbinding partner through labelling one with a detectable label andbringing it into contact with the other which has been immobilised on asolid support. Suitable detectable labels include ³⁵S-methionine whichmay be incorporated into recombinantly produced S6K1 and/or the bindingpartner thereof. The recombinantly produced S6K1 and/or binding partnermay also be expressed as a fusion protein containing an epitope whichcan be labelled with an antibody. Alternatively, double-labelling may beused as is well known in the art, for example using a radioactive labeland a scintillant.

Generally, a protein which is immobilized on a solid support may beimmobilized using an antibody against that protein bound to a solidsupport or via other technologies which are known per se. A preferred invitro interaction may utilise a fusion protein including a tag, such asglutathione-S-transferase (GST) or His6. The tag may be immobilized byaffinity interaction, for example on glutathione agarose beads orNi-matrices, respectively.

In an in vitro assay format of the type described above the putativeinhibitor compound can be assayed by determining its ability to modulatethe amount of labelled S6K1 or binding partner which binds to theimmobilized binding partner, e.g., GST-binding partner or GST-S6K1 asthe case may be. This may be determined by fractionating theglutathione-agarose beads by SDS-polyacrylamide gel electrophoresis.Alternatively, the beads may be rinsed to remove unbound protein and theamount of protein which has bound can be determined by counting theamount of label present in, for example a suitable scintillationcounter.

Alternatively an antibody attached to a solid support and directedagainst one of S6K1 or the binding partner may be used in place of GSTto attach the molecule to the solid support. Antibodies against S6K1 andits binding partners may be obtained in a variety of ways known as suchin the art. In an alternative mode, one of S6K1 and its binding partnermay be labelled with a fluorescent donor moiety and the other labelledwith an acceptor, which is capable of reducing the emission from thedonor. This allows an assay according to the invention to be conductedby fluorescence resonance energy transfer (FRET). In this mode, thefluorescence signal of the donor will be altered when S6K1 and itsbinding partner interact. The presence to a candidate inhibitor thatmodulates the interaction will increase the amount of fluorescencesignal of the donor.

FRET is a technique known per se in the art and thus the precise donorand acceptor molecules and the means by which they are linked to S6K1and its binding partner may be accomplished by reference to theliterature.

Suitable fluorescent donor moieties are those capable of transferringfluorogenic energy to another fluorogenic molecule or part of a compoundand include, but are not limited to, coumarins and related dyes such asfluoresceins, rhodols and rhodamines, resorufins, cyanine dyes, bimanes,acridines, isoindoles, dansyl dyes, aminophthalic hydrazines such asluminol and isoluminol derivatives, aminophthalimides,aminonaphthalimides, aminobenzofurans, aminoquinolines,dicyanohydroquinones, and europium and terbium complexes and relatedcompounds.

Suitable acceptors include, but are not limited to, coumarins andrelated fluorophores, xanthenes such as fluoresceins, rhodols andrhodamines, resorufins, cyanines, difluoroboradiazaindacenes, andphthalocyanines.

A preferred donor is fluorescein and preferred acceptors includerhodamine and carbocyanine. The isothiocyanate derivatives of thesefluorescein and rhodamine, available from Aldrich Chemical Company Ltd,Gillingham, Dorset, UK, may be used to label S6K1 and its bindingpartner. For attachment of carbocyanine, see for example Guo et al, J.Biol. Chem., 270; 27562-8, 1995.

Assays of the invention may also be performed in vivo. Such an assay maybe performed in any suitable host cell, e.g. a bacterial, yeast, insector mammalian host cell. Yeast and mammalian host cells are particularlysuitable. To perform such an assay in vivo, constructs capable ofexpressing S6K1 and its binding partner and a reporter gene constructmay be introduced into the cells. This may be accomplished by anysuitable technique, for example calcium phosphate precipitation orelectroporation. The constructs may be expressed transiently or asstable episomes, or integrated into the genome of the host cell.

In vivo assays may also take the form of two-hybrid assays. Two-hybridassays may be in accordance with those disclosed by Fields and Song,1989, Nature 340; 245-246. In such an assay the DNA binding domain (DBD)and the transcriptional activation domain (TAD) of the yeast GAL4transcription factor are fused to the first and second moleculesrespectively whose interaction is to be investigated. A functional GAL4transcription factor is restored only when two molecules of interestinteract. Thus, interaction of the molecules may be measured by the useof a reporter gene operably linked to a GAL4 DNA binding site, which iscapable of activating transcription of said reporter gene. Othertranscriptional activator domains may be used in place of the GAL4 TAD,for example the viral VP16 activation domain.

Irrespective of the form of assay used, they will typically be run withsuitable controls routine to those of skill in the art and is preferablyused to screen compounds that may be present in small moleculelibraries, peptide libraries, phage display libraries or natural productlibraries. Putative or actual inhibitors or other modulators may beprovided from any source which it is desired to screen, and may or maynot be naturally occurring or synthetic, and may or may not be peptidesor polypeptides (e.g. antibodies) or nucleic acids (e.g. siRNA).Preferred inhibitors most suited for therapeutic applications will besmall molecules e.g. from a combinatorial library such as are now wellknown in the art (see e.g. Newton (1997) Expert Opinion TherapeuticPatents, 7(10): 1183-1194). Preferred candidate substances may includesmall molecules such as PKC inhibitors.

A compound-induced modulation of S6K activity means that there is achange in S6K activity (enzymatic activity, signalling activity todownstream targets, promoter activity or expression) in the presence ofthe compound relative to when the compound is absent. In particular acompound induced inhibition of S6K activity is reflected by a decreasein S6K activity relative to when the compound is absent. Conversely, acompound induced activation of S6 activity is reflected by an increasein S6K activity.

Activators and inhibitors are referred to collectively herein asmodulators and preferably influence the kinase activity of S6K directly.Assays carried out using reconstituted components can be easily designedto achieve direct S6K inhibition (i.e., specific inhibition of S6Kcatalytic activity and not inhibition of the formation of active kinase,for example through the action of mTOR). Typically, S6 kinase 1 activitywill be selectively inhibited (i.e., preferentially over S6 kinase 2activity and other kinases and enzymes.), in particular when inhibitionof S6 kinase 1 signaling and treatment of insulin resistance or diabetesis desired. Alternatively, S6 kinase 2 activity will be specificallyactivated for the same purpose.

S6K inhibitors are compounds that reduce S6K activity, e.g., S6K1 orS6K2 activity. For example compounds that inhibit S6K enzymatic activitytypically bind to an ATP binding site in S6K or bind to a catalyticdomain of S6K. The compound preferentially inhibits S6K1 compared toS6K2 or other S6K isoforms, given the difference in phenotypes observedbetween S6K1 and S6K2 knock out mice. Thus, although compounds thatinhibit S6K2 or both S6K1 and S6K2 (such as rapamycin, its derivativesor other mTOR inhibitors) may be useful, the selective inhibition ofS6K1 is desirable for the treatment of insulin resistance or diabetes.Therefore, S6K1 inhibitors will typically reduce S6K1 activity by atleast 10%, more preferably 20%, 50%, 100% and 200% compared to the levelof reduction of S6K2 activity. Control assays may therefore be carriedout, for example with immunoprecipitated S6K2 and compared toimmunoprecipitated S6K1 to establish the selectivity of a modulator.

The screening methods of the invention may optionally further comprise afunctional assay, comprising detecting an effect on insulin resistance.Suitable methods are set out in examples below.

The screening methods may optionally include the step of administering apotential modulator to a non-human animal having an S6 kinase gene anddetermining whether insulin resistance is affected relative to when thecompound is absent, for example an effect on insulin sensitivity uponhigh fat feeding of the animal. The non-human animals will typically belaboratory mammals such as mice or rats and various doses can beadministered orally mixed with feed or by any other appropriate means,which may be chosen dependent on the properties of the compound, such asstability and targeted delivery. The S6 kinase gene may be from adifferent species as the laboratory mammal, for example the use of amouse comprising a human S6K gene, which replaces the mouse S6K gene,will be particularly useful to determine the effects of agents on humanS6K without using human subjects.

The potency and efficacy of compounds for inhibition of S6K1 can also beassessed using an animal model for insulin resistance and compared withS6K1 knock out animals.

Kits useful for screening such compounds may also be prepared inaccordance with the invention, and will comprise essentially S6K or afragment thereof useful for screening, and instructions. Typically theS6K polypeptide will be provided together with means for detecting S6Kactivity and at least one compound (putative agent) or other substancedescribed herein useful for carrying out the screening methods.

S6K for use in kits according to the invention may be provided in theform of a protein, for example in solution, suspension or lyophilised,or in the form of a nucleic acid sequence permitting the production ofS6K or a fragment thereof in an expression system, optionally in situ.

Compounds (e.g., putative agents) may be inorganic or organic, forexample an antibiotic, antibody, polypeptide or peptide, and aretypically isolated or purified. An “isolated” or “purified” compositionis substantially free of cellular material or other contaminatingproteins from the cell or tissue source from which it is derived, orsubstantially free from chemical precursors or other chemicals whenchemically synthesized. A polypeptide that is substantially free ofcellular material includes preparations of the polypeptide in which thepolypeptide is separated from cellular components of the cells fromwhich it is isolated, e.g., the polypeptide is recombinantly produced.Preferably, a preparation of a therapeutic compound, e. g., an S6Kinhibitor, is at least 75%, more preferably 80%, more preferably 85%,more preferably 90%, more preferably 95%, more preferably 98%, and mostpreferably 99 or 100% of the dry weight of the preparation. Mixtures ofcompounds may also be tested during initial stages of screening.

Compounds may therefore include antibodies, preferably monoclonalantibodies, that are specific for S6K, in particular S6K1, in the senseof being able to distinguish between the polypeptide it is able to bindand other polypeptides of the same species for which it has no orsubstantially no binding affinity (e.g. a binding affinity of at leastabout 1000× worse). Specific antibodies bind an epitope on the moleculethat is either not present or is not accessible on other molecules. Forexample for isoform-specific inhibition (selective inhibition of S6K1activity over S6K2 activity), the antibody may interfere with theC-terminal domain of S6K, which is not highly conserved between S6K1 andS6K2. Antibodies may be obtained using techniques which are standard inthe art. For example antibodies may be obtained from immunised animalsusing any of a variety of techniques known in the art, and screened,preferably using binding of antibody to antigen of interest. Forinstance, Western blotting techniques or immunoprecipitation may be used(Armitage et al, Nature, 357:80-82, 1992).

As an alternative or supplement to immunising a mammal with a peptide,an antibody specific for a protein may be obtained from a recombinantlyproduced library of expressed immunoglobulin variable domains, e.g.using lambda bacteriophage or filamentous bacteriophage which displayfunctional immunoglobulin binding domains on their surfaces; forinstance see WO92/01047.

Antibodies may be modified in a number of ways and include antibodyfragments, derivatives, functional equivalents and homologues ofantibodies, including synthetic molecules and molecules whose shapemimics that of an antibody enabling it to bind an antigen or epitope.Example antibody fragments, capable of binding an antigen or otherbinding partner are the Fab fragment consisting of the VL, VH, Cl andCH1 domains; the Fd fragment consisting of the VH and CH1 domains; theFv fragment consisting of the VL and VH domains of a single arm of anantibody; the dAb fragment which consists of a VH domain; isolated CDRregions and F(ab′)2 fragments, a bivalent fragment including two Fabfragments linked by a disulphide bridge at the hinge region. Singlechain Fv fragments are also included.

Humanized antibodies in which CDRs from a non-human source are graftedonto human framework regions, typically with the alteration of some ofthe framework amino acid residues, to provide antibodies which are lessimmunogenic than the parent non-human antibodies, are also includedwithin the present invention.

As is apparent to one of skill in the art, a monoclonal antibody may besubjected to the techniques of recombinant DNA technology to produceother antibodies or chimeric molecules that retain the specificity ofthe original antibody. Such techniques may involve introducing DNAencoding the immunoglobulin variable region, or the complementaritydetermining regions (CDRs), of an antibody to the constant regions, orconstant regions plus framework regions, of a different immunoglobulin.See, for instance, EP-A-184187, GB-A-2188638 or EP-A-0239400. Cloningand expression of chimeric antibodies are described in EP-A-0120694 andEP-A-0125023.

Peptides can also be used as inhibitors of S6K activity, such as asynthetic peptide containing a putative autoinhibitory domain (S6 kinase1 residues 400-432; Flowtow and Thomas, 1992) or indeed the syntheticsubstrates referred to above (e.g., S6 230-249, Ala 235), which canfurther be used to model new inhibitors.

Alternatively, S6K activity in a cell can be reduced using nucleicacids, e.g. by pre- or post-transcriptional silencing. Thus, S6Ksequences (in particular sequences specific to S6K1) may be insertedinto the vectors as described above in an antisense orientation in orderto provide for the production of antisense RNA or ribozymes.

Nucleic acid sequences selective for S6K1 over S6K2 include thoseencoding amino acids 33-77 and amino acids 454-525 (numbering refers tothat of SEQ ID NO:3 in US2003/0083284, which is hereby incorporated byreference) or portions thereof, which are typically at least 15, 18 ormore nucleotides in length. Sequence comparisons may be made essentiallyas described above using FASTA and FASTP (see Pearson & Lipman, 1988.Methods in Enzymology 183: 63-98) to establish specificity of thesequences.

An alternative to anti-sense is to use double stranded RNA (dsRNA),which has been found to be even more effective in gene silencing thanantisense alone (Fire A. et al Nature, Vol 391, (1998)). dsRNA mediatedsilencing is gene specific and is often termed RNA interference (RNAi)(See also Fire (1999) Trends Genet. 15: 358-363, Sharp (2001) Genes Dev.15: 485-490, Hammond et al. (2001) Nature Rev. Genes 2: 1110-1119 andTuschl (2001) Chem. Biochem. 2: 239-245).

RNA interference is a two-step process. First, dsRNA is cleaved withinthe cell to yield short interfering RNAs (siRNAs) of about 21-23ntlength with 5′ terminal phosphate and 3′ short overhangs (˜2 nt). ThesiRNAs target the corresponding mRNA sequence specifically fordestruction (Zamore P. D. Nature Structural Biology, 8, 9, 746-750,(2001). Thus in one embodiment, the inhibition is achieved using doublestranded RNA comprising an S6K-encoding sequence, in particular asequence selective for S6K1, which may for example be a double strandedRNA (which will be processed to siRNA, e.g., as described above).Cellular defense mechanisms, such as the PKR pathway will typically needto be circumvented, for example using siRNA directed against theindividual components. These RNA products may be synthesised in vitro,e.g., by conventional chemical synthesis methods.

However, to avoid the PKR pathway, chemically synthesized siRNA duplexesof about 21-23 nucleotides in length with 3′-overhang ends arepreferably used (Zamore P D et al Cell, 101, 25-33, (2000)). SyntheticsiRNA duplexes have been shown to specifically suppress expression ofendogenous and heterologeous genes in a wide range of mammalian celllines (Elbashir S M. et al. Nature, 411, 494-498, (2001)).

Thus, siRNA duplexes containing between 20 and 25 bps, more preferablybetween 21 and 23 bps, of the S6K sequence, in particular sequencesselective for S6K1 over S6K2, include form one aspect of the inventione.g. as produced synthetically, optionally in protected form to preventdegradation.

Alternatively siRNA may be produced from a vector, in vitro (forrecovery and use) or in vivo. Accordingly, the vector may comprise anucleic acid sequence encoding S6K (including a nucleic acid sequenceencoding a variant or fragment thereof), suitable for introducing ansiRNA into the cell in any of the ways known in the art, for example asdescribed in any of references cited herein, which references arespecifically incorporated herein by reference.

In one embodiment, the vector may comprise a nucleic acid sequenceaccording to the invention in both the sense and antisense orientation,such that when expressed as RNA the sense and antisense sections willassociate to form a double stranded RNA. This may for example be a longdouble stranded RNA (e.g., more than 23 nts), which may be processed invitro with Dicer to produce siRNAs (see for example Myers (2003) NatureBiotechnology 21:324-328) or siRNA, hairpin structures. Alternatively,the sense and antisense sequences are provided on different vectors.These vectors and RNA products are useful, for example to inhibit denovo production of the S6K polypeptide in a cell. Such nucleic acids andvectors can be introduced into a host cell or administered to a mammalin a suitable form.

Thus, nucleic acids, such as siRNA can be administered to inhibit S6K1activity. SiRNA technology can be routinely applied based on sequencesspecific for S6K1, such as AGTGTTTGACATAGACCTG or preferablyAAGGGGGCTATGGAAAGGTTT. Targeted expression of siRNAs can be achievedusing tissue-specific promoters, such as promoters specific for adiposetissue, muscle, liver or other tissues mediating insulin resistance andglucose homeostasis.

For ease of administration, however, the compound is preferably a smallmolecule, which might bind to the catalytic site or ATP binding site.For isoform-specific inhibition, the compound may interfere with theC-terminal domain of S6K, which is not highly conserved between S6K1 andS6K2.

In order to potentially improve S6K modulators, isolated S6K can be usedto establish secondary and tertiary structure of the whole protein or atleast of the areas responsible for the enzymatic activity. Conventionalmethods for the identification of the 3-dimensional structure are, forexample X-ray studies or NMR studies. The data obtained with these orcomparable methods may be used directly or indirectly for theidentification or improvement of modulators of S6K, such as to provideselectivity between S6K1 and S6K2. A commonly used method in thisrespect is, for example computer aided drug design or molecularmodelling.

Compounds according to the invention may be identified by screeningusing the techniques described hereinbefore, and prepared by extractionfrom natural or genetically modified sources according to establishedprocedures, or by synthesis, especially in the case of low molecularweight chemical compounds. Proteinaceous compounds may be prepared byexpression in recombinant expression systems, for example a baculovirussystem, or in a bacterial system. Proteinaceous compounds are mainlyuseful for research into the function of signalling pathways, althoughthey may have a therapeutic application, such as humanized inhibitoryantibodies directed against S6 kinase 1.

Low molecular weight compounds, on the other hand, are preferablyproduced by chemical synthesis according to established procedures. Theyare primarily indicated as therapeutic agents. PKC inhibitors, orderivatives or modifications thereof, may be used as potential agentseffective in selectively inhibiting S6K1 and in treating insulinresistance or diabetes. Low molecular weight compounds and organiccompounds in general may be useful as agents for use in the treatment ofinsulin resistance or diabetes.

The present invention also provides a method for reducing insulinresistance comprising contacting a cell, in particular myocytes,adipocytes and/or hepatocytes with an effective amount of an S6 kinase 1inhibitor. Thus, also provided by the invention are compounds thatdirectly modulate S6 kinase activity for use in treating insulinresistance and diabetes. In particular, compounds that selectivelyinhibit S6 kinase 1 activity over S6K2 activity (not, for example aninhibitor of mTOR, which would result in the inhibition of S6K2, whichis preferably avoided) for use in treating individuals suffering from orat risk of developing insulin resistance or diabetes are provided.

Diabetes as used herein refers to Type II diabetes resulting, forexample from obesity or overweight conditions resulting from fataccumulation, or from high circulating fatty acids. Therefore insulinresistance or diabetes resulting from high fat diets rather thanconditions resulting in reduced pancreatic beta cells are intended.

S6K modulators (e.g., inhibitors) for use in treating insulin resistanceor diabetes may be formulated as medicaments according to conventionalmethodology, depending on the exact nature of the modulator, and willtypically comprise the modulator or a precursor thereof in associationwith a biologically acceptable carrier. In considering varioustherapies, it is understood that such therapies may be targeted totissues demonstrated to express S6K1, in particular to adipose tissue,liver and muscle.

Compounds are administered at a dose that is therapeutically effective.The term “therapeutically effective amount” as used herein means thatthe amount of a compound (s) or pharmaceutical composition elicits abeneficial biological or medicinal response in a tissue, system, animalor human. For example a therapeutically effective amount of an S6K1inhibitory compound is a dose that leads to a clinically detectableimprovement in insulin resistance or diabetes.

Treatment includes the management and care of an individual for thepurpose of alleviating a symptom of insulin resistance. Treatmentincludes the administration of a compound to prevent the onset ofsymptoms or complications of the disorder, alleviating the symptoms orcomplications, or eliminating the condition or disorder.

Delivery of the modulator to the affected cells and tissues can beaccomplished using appropriate packaging or administration systems. Forexample the modulator may be formulated for therapeutic use with agentsacceptable for pharmaceutical administration and delivered to thesubject by acceptable routes to produce a desired physiological effect.An effective amount is that amount that produces the desiredphysiological effect, such as, reduced insulin resistance and type IIdiabetes.

In a further aspect of the invention, the invention also provides amodulator (e.g., selective inhibitor of S6 kinase 1) for the manufactureof a medicament for the treatment or prophylactic treatment of insulinresistance or diabetes. Suitable modulators, in particular inhibitors,such as those identified in the functional or other assays discussedabove, may be incorporated into medicaments e.g. after further testingfor toxicity. Thus the relevant methods may include the further step offormulating a selected modulator as a medicament for a disease e.g. inwhich it is desired to control insulin resistance or diabetes. Suchinhibitors and medicaments for use in the treatment of these diseases,and methods of treatment comprising their use form further aspects ofthe invention.

The compositions may include, in addition to the above constituents,pharmaceutically-acceptable excipients, preserving agents, solubilizers,viscosity-increasing substances, stabilising agents, wetting agents,emulsifying agents, sweetening agents, colouring agents, flavouringagents, salts for varying the osmotic pressure, buffers, or coatingagents. Such materials should be non-toxic and should not interfere withthe efficacy of the active ingredient. The precise nature of the carrieror other material may depend on the route of administration. Examples oftechniques and protocols can be found in “Remington's PharmaceuticalSciences”, 16th edition, Osol, A. (ed.), 1980.

Where the composition is formulated into a pharmaceutical composition,the administration thereof can be effected parentally such as orally,nasally (e.g. in the form of nasal sprays) or rectally (e.g. in the formof suppositories). However, the administration can also be effectedparentally such as intramuscularly, intravenously, cutaneously,subcutaneously, or intraperitoneally (e.g. in the form of injectionsolutions).

Thus, for example where the pharmaceutical composition is in the form ofa tablet, it may include a solid carrier such as gelatine or anadjuvant. For the manufacture of tablets, coated tablets, dragees andhard gelatine capsules, the active compounds and theirpharmaceutically-acceptable acid addition salts can be processed withpharmaceutically inert, inorganic or organic excipients. Lactose, maize,starch or derivatives thereof, talc, stearic acid or its salts etc. canbe used, for example as such excipients for tablets, dragees and hardgelatine capsules. Suitable excipients for soft gelatine capsules are,for example vegetable oils, waxes, fats, semi-solid and liquid polyolsetc. Where the composition is in the form of a liquid pharmaceuticalformulation, it will generally include a liquid carrier such as water,petroleum, animal or vegetable oils, mineral oil or synthetic oil.Physiological saline solution, dextrose or other saccharide solution orglycols such as ethylene glycol, propylene glycol or polyethylene glycolmay also be included. Other suitable excipients for the manufacture ofsolutions and syrups are, for example water, polyols, saccharose, invertsugar, glucose, trihalose, etc. Suitable excipients for injectionsolutions are, for example water, alcohols, polyols, glycerol, vegetableoils, etc. For intravenous, cutaneous or subcutaneous injection, orintracatheter infusion into the brain, the active ingredient will be inthe form of a parenterally-acceptable aqueous solution which ispyrogen-free and has suitable pH, isotonicity and stability. Those ofrelevant skill in the art are well able to prepare suitable solutionsusing, for example isotonic vehicles such as Sodium Chloride Injection,Ringer's Injection, Lactated Ringer's Injection. Preservatives,stabilisers, buffers and/or other additives may be included, asrequired.

Also provided are methods of evaluating treatments for diabetes andinsulin resistance, the method comprising administering a therapeuticagent (for example identified by the screening methods provided herein)to a non-human animal comprising an S6 kinase gene, in particular S6K1,and determining the effect of the agent on insulin resistance.Alternatively, a sample, such as adipose, muscle or liver tissue, orperipheral blood can be withdrawn and tested for S6K levels or activitylevels to establish a modulatory effect on S6K.

In addition, methods of evaluating treatments for insulin resistance ordiabetes, comprising administering a potential therapeutic agent to anon-human animal deficient in S6 kinase (such as a knock-out animal), inparticular deficient in S6K1, and determining the effect of the agent.Such methods can be used to establish whether any unwanted side effectsof the identified therapeutic agent are present.

The present inventors have shown that wild type mice fed a high fat diethave strikingly elevated S6K1 activity (see example 9). The inventiontherefore also provides a method of diagnosing a predisposition toinsulin resistance or diabetes, comprising: obtaining a sample from anindividual, detecting the level of S6 kinase, preferably S6 kinase 1, inthe sample and correlating a change in the amount of S6 kinase in thesample when compared to a normal control value or range of values with apredisposition to insulin resistance or diabetes. The presence of S6kinase can be easily determined using antibodies or using activityassays as described above. S6K expression can also be detected at thetranscript level, e.g., using PCR techniques. When protein levels aredetected, antibodies specific for S6K2 can be used as a control whenS6K1 specific measurements are desired. In general, an increase in S6kinase 1 activity of at least 10%, preferably at least 20%, 30%, 40% or50% when compared to a normal control value or range of values isindicative of a predisposition to insulin resistance or diabetes. Mostpreferably, the increase in activity is at least 2-fold than that of acontrol value. The sample may be any tissue sample or body fluid, but ispreferably adipose, muscle or liver tissue.

To determine the kinase activity of S6K 1 (independent of other S6Kisoforms such as S6K2), a sample is obtained from a test subject. Cellspresent in the sample can be lysed and proteins extracted. Optionally,further purification steps can be carried out. The sample can then besubjected to immunoprecipitation using an S6K1 specific antibody, whichare known in the art. Following immunoprecipitation of S6K1, a standardkinase assay is performed as described above.

The invention is further described, for the purposes of illustrationonly, in the following examples.

EXAMPLES

Methods of molecular genetics, protein and peptide biochemistry andimmunology referred to but not explicitly described in this disclosureand examples are reported in the scientific literature and are wellknown to those skilled in the art. For example standard methods ingenetic engineering are carried out essentially as described in Sambrooket al., Molecular Cloning: A laboratory manual, 2nd Ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor N.Y., 1989.

EXAMPLE 1 S6K1 Deficient Mice are Smaller than Wild Type

S6K1 deficient mice were previously shown to have a reduction in bodysize during embryogenesis but the effect was thought to be mostlyovercome by adulthood, diminishing from 20 to 15% by 11 weeks of age.This example demonstrates that as they age, S6K1 deficient mice maintaina lower body weight relative to wild type mice. Male mice on a normalchow diet (NCD, 4% of total calories derived from fat, 3035 kcal kg-1,KLIBA-NAFAG, Switzerland) were followed over a period of seventeen weeksfrom ten weeks of age.

S6K1 deficient mice were generated as described by Shima et al. (1998,EMBO J., 17, 6649-6659). Mice were kept on a hybrid background derivedfrom the C57BI/6 and 129Oia mouse strains, housed in groups of 12 (incages of 3) and maintained on a 12 hour light/12 hour dark cycle (lightson at 06:00 GMT). Body weight was recorded weekly in wild type (wt) andS6K1 deficient mice fed regular chow diet. Unexpectedly, the resultsshowed that the rate at which S6K1−/− mice gained weight was much slowerthan that of wild mice, such that the difference in weight attwenty-seven weeks, as compared to ten weeks, had increased to 25%. Itshould also be noted S6K1−/− mice displayed little variation in weightas compared to wild type (wt) mice.

EXAMPLE 2 S6K1 Deficient Mice have Reduced Body Fat

Mice were dissected to determine the cause of the lower body weightexhibited by S6K1 deficient mice described in example 1. S6K1 deficientmice were shown to have less intra-abdominal fat pads. Each fat mass andorgan mass was weighed from tissue removed from 6-month old male mice.Dissection of S6K1 mice revealed a severe reduction of epididymal whiteadipose tissue fat relative to wild type mice (0.8% +/−0.1% comparedwith 3.4% +/−0.1%; the values are averaged values +/−standard errormean; S.E.M). Percentage brown adipose tissue/body weight was alsoreduced in S6K1 deficient mice (0.5% +/−0.05% compared with 1.0+/−0.1%),whereas organ size was essentially unaffected. Similar results werefound also in female mice. The decrease in fat was not associated with aselective decrease in the deposition of fat in liver or muscles.

The results establish that S6K1−/− mice have reduced body white fat andbrown fat relative to wild type mice.

EXAMPLE 3 S6K1 Deficient Adipocytes are Smaller

To establish why S6K1 deficient mice exhibit less fat, adipose tissuesections were stained with hematoxylin and eosin and visualized by20-fold magnification using a histology microscope. Both epididymalwhite adipose tissue (WAT) and brown adipose tissue from S6K1 deficientmice exhibited smaller cell size when compared to wild type.

Analysis of the size of fat cells in epididymal fat pads by eitherscanning electron microscopy or by hematoxyline-eosine staining showed astriking reduction in cell size, with quantitation revealing thatadipocytes from S6K1−/− mice were 71% smaller than those of wild typemice (wt: 3129±904, n=3; S6K1−/−: 918±189 mm2 , n=3, P<0.05), with manyadipocytes exhibiting a multilocular phenotype. These studies alsorevealed that the distribution of cell size, like body weight, was muchmore homogeneous in S6K1−/− mice as compared to wild type mice and arough calculation of cell surface areas versus total mass suggested thatthere was little effect on cell number. In summary, results fromelectron microscopy, histology and cell density/size analysisestablishes that the reduction in fat in S6K1 deficient animals is dueto a reduction in fat cell size.

EXAMPLE 4 Diet is Not the Cause of Less Fat in S6K1 Deficient Mice

To establish whether S6K1 mice exhibited dietary differences over wildtype mice, which would explain the reduction in fat in S6K1 mice, foodintake per mouse was measured every other day for 15 days using normalor high fat chow. Irrespective of whether placed on normal or high fatdiet, S6K1 deficient mice eat the same total amount of food as wild type(about 4.6+/−0.1 g food/mouse/day), but compared to body weight, theyeat much more (about 17% or more, or 0.18 compared to 0.15 g food/g bodyweight/day).

Furthermore, despite their apparent leanness, the mice did not appear tobe starving as glucose homeostasis appeared normal (Table I below),consistent with the fact that there was no increase in ketone bodyformation (D-β-hydroxybutyrate values 1.3 mg/dl±0.05 for wild type mice,n=8; 1.4 mg/dl±0.1 for S6K1^(−/−) mice, n=7; P=0.8).

EXAMPLE 5 S6K1 Deficient Mice Exhibit an Enhanced Metabolic Rate

The increased food uptake, combined with reduced WAT, raised thepossibility of enhanced metabolic activity. The metabolic rate of S6K1deficient and wild type mice were examined by indirect calorimetry tomonitor oxygen consumption and carbon dioxide production every 15 minover an eight-hour fasting period employing an Oxymax (ColumbusInstruments, Columbus, Ohio).

The results show a striking 27% increase in the rate of oxygenconsumption in S6K1−/− mice versus wild type mice through out theexperiment. Calculation of the respiratory exchange ratio (RER=ratio ofCO2 produced to O2 consumed) gave values of 0.713±0.004 for wild typemice and 0.709±0.003 for S6K1−/− mice; P<0.01), arguing that bothanimals were largely utilizing fatty acids as an energy source.

Metabolite assays were carried out as follows. Blood was collected fromretroorbital sinus after overnight fast or 1 hr after beginning of themeal followed by overnight fast. Nonesterified fatty acids, andtriglycerides were determined by enzymatic assays (Boehringer-Mannheim,Germany). Plasma leptin was measured using Rat leptin RIA kit (LincoResearch, St Louis, Mo.). The results are shown in Table I. Plasmaleptin levels were significantly reduced in S6K1−/− mice (Table I) inkeeping with the mice's increased consumption of food relative to bodyweight. TABLE I one-hour postprandial insulin, triglycerides, free fattyacids and leptin levels after overnight fasting of 6 month old male wildtype, and S6K1 deficient mice fed a normal, or high fat diet. WT S6K1−/− Diet Normal High Fat Normal High Fat Insulin (μg/l) 0.57 ± 0.09 1.91± 0.78 0.38 ± 0.08* 0.22 ± 0.06* Triglycerides 0.61 ± 0.09 1.08 ± 0.150.55 ± 0.06 0.95 ± 0.14 (mmol/l) Free fatty acids 0.26 ± 0.05 0.27 ±0.02 0.19 ± 0.04 0.56 ± 0.10* (mmol/l) leptin (ng/ml) 5.50 ± 0.82 12.6 ±0.00 3.42 ± 0.75 6.34 ± 2.35*Data represent means ± s.e.m.*P < 0.05 compared with wild type (n = 6 to 18).P values were calculated by two-tailed, unpaired Student's t-test.

Although not wishing to be bound by theory, given the reduced adiposetissue mass, increased metabolic rate, and the fact that when correctedfor body weight, plasma triglycerides and free fatty acids were similarbetween genotypes (Table I), it was reasoned that in the absence ofS6K1, either triglycerides in adipose tissue were being rapidly utilizedor that the free fatty acids never reached adipose tissue for storage,but were immediately taken up by muscle and oxidized.

Although WAT is not normally an energy consuming tissue, that there wasno difference in the basal levels of circulating fatty acids in S6K1−/−mice, as compared to wild type mice (Table I), suggested that free fattyacids may be directly oxidized in WAT. Consistent with this hypothesis,electron micrographs revealed the presence of many multilocularadipocytes, which displayed a dramatic increase in the size and numberof mitochondria, phenotypes completely absent in adipocytes from wildtype mice. Others have shown that overexpression of UCPI in WAT inducesa similar phenotype, and UCP1 levels, measured by quantitative realtime-PCR, were dramatically upregulated in WAT from S6K1−/− mice ascompared to WAT from wild type mice.

EXAMPLE 6 S6K1 Deficient Mice Exhibit Increased Basal Lipolysis

The breakdown of triglycerides in adipose tissue (lipolysis) wasmeasured by monitoring the release of either fatty acids or glycerolfrom mature adipocytes. Primary adipocytes were prepared from epididymalfat pads by collagenase digestion as described previously (Marette etal., 1991). Cells were incubated for 30 min at 37° C. with or withoutnorepinephrine (Sigma-Aldrich SARL, St-Quentin Fallavier, France) atdifferent concentrations (10⁻⁸ to 10⁻⁵ M). Norepinepherine is abeta-adrenergic agonist and stimulates the breakdown of adipocytetriglyceride to glycerol and free fatty acids (lipolysis) and increasesthe basal metabolic rate (thermogenesis). Despite the reduction in cellsize of epididymal adipocytes (see example 3), the results showed thatthe basal rate of fatty acid release was approximately 5-fold higher inadipocytes from S6K1−/− mice as compared to wild type mice. The rate offatty acid release increased in both genotypes in a dose dependentmanner upon the addition of norepinephrine, reaching similar maximumvalues, with the increase much steeper in wild type mice. Similarresults were obtained for the release of glycerol. Therefore, S6K1−/−mice are protected against fat accumulation partly due to a sharpincrease in basal lipolysis.

EXAMPLE 7 S6K1 Deficient Mice Exhibit Impaired Adipogenesis

During the isolation of mature adipocytes for the lipolysis studies, itbecame evident that there were few preadipocytes present in epididymalWAT of S6K1−/− mice. This, along with the inability of adipocytes tostore triglycerides, prompted experiments to compare the ability ofmouse embryonic fibroblasts (MEFs) from S6K1−/− and wild type embryos todifferentiate into adipocytes using an adipocyte differentiation mix.

Briefly, adipocyte differentiation was induced essentially as previouslydescribed (Hansen et al., 1999, J. Biol. Chem., 274, 2386-2393) usingwild type or S6K1 deficient mouse embryonic fibroblasts (MEFs). Thepassage number of MEFs was within one passage. For differentiation,2-day post confluent cells (day 0) were treated with growth mediumcontaining 1 μM dexamethasone (Sigma), 0.5 mM methylisobutylxanthine(Aldrich), 5 μg/ml insulin (Boehringer Mannheim), and Ciglitazone(thiazolinedione, PPAR agonist: BIOMOL, GR-205, 0.5 μM) for 2 days. Fromday 2, the medium contained 5 μg/ml insulin and Ciglitazone and renewedevery other day. Oil red O staining: Oil red O staining solution (0.5%Oil red O in isopropyl alcohol solution-distilled water (60:40) wasfiltered. Cells were washed with PBS and stained for 30 min and thenwashed with distilled water two times. Cells deficient in S6K1 showedmuch less staining. Therefore, MEFs lacking S6K1 had a reducedadipogenic potential, as evaluated by the reduced Oil Red O lipidstaining, consistent with lower levels of aP2 mRNA. Taken together theresults suggest that S6K1−/− mice have reduced WAT because of impairedadipogenesis and an inability to store fat.

EXAMPLE 8 S6K1 Deficient Mice are Protected Against Diet-induced Obesity

The failure of S6K1−/− mice to accumulate fat with age combined withtheir overall increase in metabolic rate suggested that they may beprotected against diet-induced obesity. In this example body weight wasrecorded weekly in wild type and S6K1 deficient mice fed high fat diets(HFD, 60% of total calories derived from fat, 4057 kcal kg-1, Researchdiets, USA). When S6K1 mice are placed on a high fat diet, absolute bodyweight gain of S6K1 deficient mice was about 10.5 g over the period ofhigh fat diet feeding (from week 7 to 27 of age) compared to about 14.49 in wild type mice.

Similar to the situation on the NCD (example 1), S6K −/− mice displayedlittle variation in weight on a HFD as compared to wild type mice. Giventhe smaller body size of the knockout, relative weight gain was similarbetween genotypes (58.9% increase of body weight in S6K1 deficient micecompared to 58.0% increase of body weight in wild type after high fatdiet feeding for 5 months. Even though the relative percentage of bodyweight gain in S6K1 deficient mice was similar to wild type, they failto put on fat to the same extent as wild type mice. Over a three-monthperiod, wild type mice gained 0.1 g/g compared with 0.02 g/g fat/bodyweight by S6K1 deficient mice.

Mice of both genotypes consumed less food on a HFD than on a NCD,probably due to the higher caloric density of the HFD as compared to theNCD. Although in absolute terms, S6K1−/− mice consume the same amount offood as wild type mice, when normalized to body weight they consume 44%more food. Thus, even though S6K1 mice eat more, they do not put on fatto the same degree as wild type mice.

Indirect calorimetry measurements were conducted over an eight-hourfasting period on HFD and NCD mice. In both genotypes oxygen consumptionincreased on the HFD, as compared to the NCD, but the effect was morepronounced for S6K1−/− mice, such that the difference between S6K1−/−mice and wild type mice increased from 25% to 30%. The data furthershowed that the RER remained unchanged in S6K1−/− mice on a HFD versus aNCD, 0.708±0.002 vs 0.709±0.004, respectively, whereas in wild type micethe RER increased from 0.713±0.004 on NCD to 0.729±0.002 (n=6, P<0.01)on a HFD diet, indicative of an increase in carbohydrate relative tofatty acid oxidation. Despite the fact that S6K1−/− mice on either dietdisplayed a high metabolic rate, on a HFD they exhibited a threefoldincrease in circulating free fatty levels, whereas in wild type micethere was no significant change in free fatty acids levels (Table I).Hence, S6K −/− mice fail to accumulate fat at an appreciable rate whenchallenged with a HFD.

EXAMPLE 9 Obese Animals and Wild Type Animals on High Fat Diet ExhibitElevated S6K1 Phosphorylation in Adipose Tissue

To examine whether S6K1 is affected in adipose tissue from normal andobese genetic models, the phosphorylation of S6K1 was detected. This caneasily be carried out using phospho-specific antibodies. The level ofS6K1 T389 and S6 S240/S244 phosphorylation in adipose tissue of wildtype mice fasted for a short period was determined. Upon growth factorstimulation, S6 is multiply phosphorylated at the carboxy terminus onfive serine residues in an ordered fashion beginning with Ser236,followed sequentially by >Ser235 >Ser240 >Ser244 and Ser247. Basalvalues of S6K1 T389 and S6 S240/S244 phosphorylation are low in micefasted for a short period after being maintained on a NCD. In sharpcontrast, the same mice maintained on a HFD and treated under theidentical conditions, maintained high-elevated levels of S6K1 T389 andS6 S240/S244 phosphorylation.

The present inventors also examined S6K1 activity in ob/ob mice, agenetic model for obesity. The results show that the ob/ob micemaintained on NCD have elevated S6K1 T389 and S6 S240/S244phosphorylation as compared to wild type mice on a NCD. Preliminaryhuman data are consistent with these data and provide further supportfor S6K1 as a promising drug target in the treatment of patientssuffering from obesity and as a potential diagnostic marker.

EXAMPLE 10 Mature S6K1 Deficient Mice do not Exhibit Insulin Resistance

S6K1 deficient mice have previously been suggested to be hypersensitiveto insulin in their peripheral tissues, because they maintain normalfasting glucose levels, despite their innate hypoinsulinemia and mildglucose intolerance (Pende et al., 2000).

Glucose tolerance tests and in vivo insulin secretion were performed aspreviously described (Pende et al., 2000). Insulin tolerance tests wereperformed by intraperitoneal injection of 0.75 U/kg body weight insulinafter 3-h fast. Blood was collected before injection and 15, 30, 60 and90 min after injection.

On a Normal Chow Diet, more mature S6K1 deficient mice exhibit a slighttendency towards increased insulin sensitivity versus wild type mice, asindicated by the moderately faster rate of glucose clearance uponinsulin tolerance testing. However, the effect is small. This raised thelikelihood that S6K1 deficient mice fed on a High Fat Diet would likewild type mice become insulin resistant if fed on a High Fat Diet.

Unexpectedly, the results of such an analysis show, that despite a sharpincrease in free fatty acids (Table 1), which is implicated in theetiology of insulin resistance, S6K1 deficient mice remain insulinsensitive, whereas wild type mice display strong insulin resistance asexpected on a high fat diet. Consistent with this, wild type mice becomeglucose intolerant on a high fat diet as compared to a match set of miceon a normal chow diet. However, S6K1 deficient mice on high fat dietremain glucose tolerant, despite a further significant decrease inpostprandial (1 h) circulating insulin levels (Table1).

EXAMPLE 11 Mechanism of Insulin Sensitivity in S6K1 Deficient Mice onHigh Fat Diet

This example addresses how S6K1 deficient mice remain hypersensitive toinsulin in their peripheral tissues on a high fat diet.

After a 6 hour fast, mice were anesthetized and 0.75 Ukg-1 insulin (EliLilly) or an equal volume of vehicle was administered by i.v. injection.Liver, adipose (Epididymal fat pads) and muscle (Gastrocnemius) werecollected in liquid nitrogen 5 minutes after injection. Tyrosinephosphorylation of insulin receptor was measured in liver. Proteinextracts (1 mg) from tissue samples were prepared forimmunoprecipitation and analyzed as described (Hirosumi, 2002).Antibodies were purchased from Santa Cruz (anti-insulin receptor β),Upstate Biotechnology (anti-phosphotyrosine) and Cell Signaling(anti-PKB, anti-phospho PKB-Ser473, anti-phosphoS6K-Thr 389,anti-phospho S6 240/244, and Upstate Biotechnology(anti-phosphotyrosine).

Examination of PKB activity in adipose tissue following insulininjection, as a reporter for the insulin signaling pathway, revealedthat the activation of the kinase was suppressed in wild type micemaintained on a high fat diet versus mice raised on a normal chow diet.However, in contrast to wild type mice, there was no significantdifference in PKB activation in S6K1 deficient mice, regardless of thediet. The absence of an effect of high fat diet on PKB activation inS6K1 ko mice was also true for liver and muscle.

Examination of insulin receptor autophosphorylation shows that it isalso strongly suppressed by high fat diet in the liver of wild typemice, but not in S6K1−/− mice, raising the possibility that the insulinreceptor may be the target of the negative feedback loop.

Given these findings, it raised the possibility that on a high fat diet,S6K1 activity is elevated and that this enhanced activity is responsiblefor inducing insulin resistance. To test this possibility the presentinventors examined the level of S6K1 T389 phosphorylation and S6phosphorylation in fat and muscle, five minutes following an intravenousadministration of insulin. The results show that basal values of S6K1T389 phosphorylation are low in both high fat diet and normal chow dietanimals, but in contrast to PKB Ser 473 phosphorylation, insulinstimulates these levels even higher, potentially suppressing insulinsignalling further.

These findings raised the possibility that a potential mechanism bywhich obese humans become insulin resistant is through nutrient inducedS6K1 activation, suppressing insulin signaling through a negativefeedback loop. To test this possibility the present inventors examinedS6K1 activity in patients, which were clinically lean, obese or obeseand diabetic following a six-hour fasting period. The results show thatthe obese and the obese/diabetic patients have elevated S6K1 levels ascompared to lean patients, consistent with the feedback loop in whichS6K1 is a major negative effector in insulin signaling.

The results taken together strongly suggest that S6K1 may be a potentialtarget for drug intervention in the treatment of patients suffering fromobesity-induced diabetes or patients suffering from insulin resistancein their peripheral tissues.

The disclosure of any publication referred to herein, as well as GBapplication GB0224338.4 filed Oct. 18, 2002 is hereby specificallyincorporated by reference.

1. A method of identifying an agent effective in treating insulinresistance, said method comprising the steps of: i) incubating S6 kinasewith a compound; ii) detecting S6 kinase activity; and iii) determininga compound-induced modulation in the S6 kinase activity relative to whensaid compound is absent, wherein an alteration of the S6 kinase activityin the presence of the compound is indicative of an agent effective intreating insulin resistance.
 2. The method according to claim 1, whereinsaid modulation is inhibition of S6 kinase 1 activity.
 3. The methodaccording to claim 1, wherein said modulation is activation of S6 kinase2 activity.
 4. The method of claim 1, comprising determining S6 kinaseactivity using S6 as a substrate.
 5. The method of claim 1, comprisingdetermining S6 kinase activity using a peptide as a substrate.
 6. Amethod of screening for an agent effective in treating insulinresistance, the method comprising (a) contacting transcriptionallyactive cellular components with a nucleic acid encoding an S6K geneoperably linked to a promoter sequence or an S6K promoter sequenceoperably linked to a reporter gene in the presence of at least onecompound; and (b) detecting an effect of said compound on S6 kinaseexpression or S6 kinase promoter activity, wherein detection of amodulation in S6 kinase expression or promoter activity is indicative ofan agent effective in treating insulin resistance.
 7. The method ofclaim 6, wherein said transcriptionally active cellular components andsaid nucleic acid is present in a cell.
 8. The method of claim 6,wherein said S6 kinase is S6 kinase 1 and said modulation is a decreasein S6 kinase expression or promoter activity.
 9. The method of claim 6,further comprising detecting an effect of said agent on insulinresistance.
 10. An agent identified by claim
 6. 11. A method forreducing insulin resistance, said method comprising contacting anadipocyte, myocyte or hepatocyte with an effective amount of an S6kinase 1 inhibitor.
 12. The method of claim 11, wherein said S6K1inhibitor preferentially reduces enzymatic activity of S6K1 compared toS6K2.
 13. A method for treating or preventing the development of insulinresistance or diabetes, comprising administering to a subject apharmaceutically effective amount of an S6 kinase modulator.
 14. Themethod of claim 13, wherein said S6 modulator is an inhibitor thatpreferentially reduces S6K1 activity compared to S6K2.
 15. The method ofclaim 13, wherein said inhibitor binds to an ATP binding site in S6K1.16. The method of claim 13, wherein said inhibitor binds to a catalyticdomain of S6K1.
 17. The method of claim 13, wherein said inhibitor is anantibody or antibody fragment specific for S6 kinase
 1. 18. The methodof claim 13, wherein said inhibitor is an antisense, ribozyme or siRNAthat preferentially reduces expression of S6 kinase 1 compared to S6kinase
 2. 19. A method of diagnosing insulin resistance or apredisposition to insulin resistance, comprising: (a) detecting thelevel of S6 kinase activity in a sample from a mammal; and (b)correlating a change in S6 kinase activity when compared to a normalcontrol value or range of values with insulin resistance or apredisposition to insulin resistance.
 20. The method of claim 19,wherein said S6 kinase activity is S6K1 enzymatic activity.
 21. Themethod of claim 19, wherein an increase in the level of S6K1 activitycompared to a normal control indicates that said mammal is sufferingfrom or has a predisposition to developing insulin resistance.